In message <9305142052.AA14505 at emoryu1.cc.emory.edu> writes:
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> To: proteins at net.bio.net> From: ez027854 at othello.ucdavis.edu> Subject: C-terminal Analysis Protocol
> Date: 14 May 93 19:24:22 GMT
> Sender: Mike.Oda at net.bio.net (ez027854 at hamlet.ucdavis.edu)
>> Hi,
> I have recently cloned a c-terminally processed protein and am in
> the process of analysing both the precursor molecule and the processed
> product. I am wondering if anyone out there could assist me by sending
> me a protocol for c-terminal analysis of a peptide. My searches have
> lead me to relatively "old" protocols from the early 80s and I am wondering
> if there has been any recent advances in this area that I may have
> overlooked.
> The reason I ask is because the results obtained from the use of the protocol
> I have (exopeptidase, time point cleavage) gives rather dirty results that
> are nearly unusable beyond two amino acids.
> Any help in this area would be greatly appreciated.
> Thanks
> Michael Oda
> (ez027854 at hamlet.ucdavis.edu)
>> :: C-terminal Analysis Protocol
The last time I looked into this, the exopeptidase approach was the only one
that worked very well, and as you have discovered, in many cases the kinetic
approach breaks down fairly quickly.
Consider analyzing your reaction products by electrospray mass spectrosopy.
Since you know the sequence, this can provide you with the site of cleavage with
great accuracy and doesn't require too much material. Post-translational
modifications of the precursor molecule should be identified by this proceedure
also.
Keith D. Wilkinson genekdw at emoryu1.cc.emory.edu
Department of Biochemistry grdbckdw at emuvm1.cc.emory.edu
Emory University School of Medicine Voice (404) 727-5980
Atlanta, GA 30322 Fax (404) 727-2738
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